Targeting altered tumor cell metabolism might provide an attractive opportunity for patients with acute myeloid leukemia (AML). An amino acid dropout screen on primary leukemic stem cells and progenitor populations revealed a number of amino acid dependencies, of which methionine was one of the strongest. By using various metabolite rescue experiments, NMR-based metabolite quantifications and 13C-tracing, polysomal profiling, and ChIP-seq, we identified that methionine is used predominantly for protein translation and to provide methyl groups to histones via S-adenosylmethionine for epigenetic marking. H3K36me3 was consistently the most heavily impacted mark following loss of methionine. Methionine depletion also reduced total RNA levels, enhanced apoptosis and induced a cell cycle block. ROS levels were not increased following methionine depletion and replacement of methionine with glutathione or N-acetylcysteine could not rescue phenotypes, excluding a role for methionine in controlling redox balance control in AML. Although considered to be an essential amino acid, methionine can be recycled from homocysteine. We uncovered that this is primarily performed by the enzyme methionine synthase and only when methionine availability becomes limiting. In vivo, dietary methionine starvation was not only tolerated by mice, but also significantly delayed both cell line and patient-derived AML progression. Finally, we show that inhibition of the H3K36-specific methyltransferase SETD2 phenocopies much of the cytotoxic effects of methionine depletion, providing a more targeted therapeutic approach. In conclusion, we show that methionine depletion is a vulnerability in AML that can be exploited therapeutically, and we provide mechanistic insight into how cells metabolize and recycle methionine.
With immune therapies on the rise, an in-depth understanding of the immunological changes in leukemic bone marrow (BM) niches becomes indispensable. Being an crucial part of the tumor microenvironment (TME) in solid tumours, tumour-associated macrophages are often associated with poor prognosis (Bruni et al. 2020). Yet, in acute myeloid leukaemia (AML) the role of macrophages has not been thoroughly studied. The expression of the M2-markers CD163 and CD206 in the AML BM cell population predicted poor clinical outcome. We identified that this expression emerges from a more mature (CD45 midSSC highHLA-DR +CD14 +CD16 +/-) myeloid cell population (hereafter called AML-associated macrophages - AAM) and not from the leukemic blasts. By employing flow cytometry analysis (FACS) we noted a decrease in the expression of the M1-marker (CD80) and an increase of the M2-markers CD163/CD206on AAM (n=70) compared to healthy donors (HD, n=10). Unsupervised clustering based on the CD163/CD206 levels detected on AAM generated 4 distinct clusters, whereby patients within the CD163 low/CD206 low cluster displayed better overall survival than the other clusters. In vitro, the co-culture of HD-derived M1 macrophages and AML primary/cell lines reduced AML growth via apoptosis induction and cell cycle arrest, while M2-macrophages promoted AML survival and phagocytosis/drug-resistance when treated with FLT3/BCL2 inhibitors. Primary AML cells were also able to repolarize M1- into M2-macrophages, suggesting that leukemic cells actively remodel their microenvironment. Next, we evaluated the impact of M2-macrophages on leukemogenesis in a patient derived xenograft (PDX) model, using the notoriously difficult to engraft primary Acute Promyelocytic Leukaemia (APL) cells (n=7 patient samples). Intra-BM injection of M2-macrophages and retro-orbital transplant of primary APL cells induced full-blown APL in NSGS mice. More strikingly, ex vivo culture of APL cells on M2-macrophages (48h) was sufficient to "train" these cells to engraft and induce fatal APL. Maintenance of self-renewal was shown in a secondary transplant and an enhanced frequency of leukemic stem cells was assessed by in vivo LTC-IC assays. To identify the biological changes acquired by leukemic blasts, we performed RNA sequencing comparing AML/APL samples at diagnosis to cells that were "trained" (48 h) on M2-macrophages or on MS5 mesenchymal BM stromal cells. Gene ontology and gene set enrichment analysis on the genes up-regulated upon M2 co-culture were significantly enriched for cell migration, cell cycle progression and oxidative phosphorylation (OXPHOS) signatures. In line with our RNAseq data, we noted improved in vivo homing of primary APL cells to the BM within 18 h post-transplant upon ex vivo M2 co-culture compared to diagnosis (n=7 APL blasts). Concurrently, we detected increased levels of surface protein expression Integrin-α4 (CD49d) and -α5 (CD49e) on APL/AML blast cells after M2 exposure. The CD49d expression remained high in primary and secondary transplants. Using seahorse measurements, we confirmed the increased respiration capacity (basal and maximum) of primary AML/APL cells (n=7) after exposure to M2 macrophages compared to MS5.FACS analysis revealed that M2-macrophages were able to transfer more mitochondria than MS5 cells to primary AML cells, which could underlie the observed increase in OXPHOS mitochondrial metabolism. Treatment with Etomoxir (50 µM), prevented the gain in functional respiration when AML blast were co-cultured on M2-macrophages, while no changes were observed for MS5 co-cultures, suggesting increased fatty acid oxidation to drive the OXPHO-like state. Finally, we noted that training on M2 macrophages significantly increased colony formation and endowed the cells with long term proliferation in liquid cultures for over 30 days. Overall, we reveal that the frequency of M2-macrophages is up-regulated in a subgroup of AML patients representing a group with poor prognosis. M2 macrophages can support leukemic growth and therapy-resistance, and support fatal APL in PDX models. Even an in vitro exposure to M2 macrophages suffices to alter adhesion, homing and metabolic characteristics of leukemic blasts to allow efficient engraftment and fatal leukemogenesis. Our study uncovers how the TME can contribute to leukemic transformation which provides alternative avenues for therapeutic interventions. Disclosures Silveira: BMS/Celgene: Research Funding; Servier/Agios: Research Funding; Abbvie: Speakers Bureau; Astellas: Speakers Bureau. Quek: BMS/Celgene: Research Funding; Servier/Agios: Research Funding. Mota: Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Technopharma: Speakers Bureau; Bristol Myer Squibb: Speakers Bureau; Bayer: Speakers Bureau; Pfizer: Speakers Bureau; AstraZeneca: Speakers Bureau; Astellas: Speakers Bureau; Ipsen: Speakers Bureau; Amgen: Speakers Bureau.
Aims: The active methylation of histones plays an important role in regulation of gene expression. Nuclear SET Domain (NSD) Histone Lysine Methyltransferases family (KMT) is composed of three members: NSD1, 2 and 3 which regulates gene expression through methylation of H3K36. NSD2 overexpression, initially reported in multiple myeloma, was recently associate with EZH2 mRNA levels through interaction with H3K27me3 which suppresses the expression of miR-203, miR-26a and miR-31 leading to the up regulation of NSD2 mRNA levels. This correlation has been observed in various types of cancer, although there is no data in myeloid malignancies. Aim: To evaluate the gene expression profile of the NSD family in three de novo AML cohorts: one Brazilian cohort and two public databases addressing its applicability in prediction of treatment outcomes, retrospectively. Methods: As a learning set, samples from 146 subjects (age, 18-87y) were analyzed. Seventy (48%) patients were treated in a University Hospital that serves as reference for northeast of Brazil, while 76 patients were treated in a center of similar characteristics in the southeast of the country. The baseline features distribution was similar between centers. The treatment protocol was described elsewhere (Lima AS et al., Blood, 2015), but was based on anthracycline and cytarabine for induction and intermediate to high doses of cytarabine as consolidation. As controls, sixteen samples of CD34+ cells isolated from total bone marrow (BM) of healthy volunteers (age, 18-60y) were analyzed. The external validation cohort composed by 173 patients (age, 18-88y) were obtained from TCGA AML study available online on CBioPortal for Cancer Genomics (Gao et al. 2013). Patients were dichotomized into "low" and "high" NSD1, 2 and 3 expression groups based on survival receiver operating characteristic (ROC) curve and the C index analyses. To validate our data, NSD transcript levels from an independent cohort was used (525 patients from Amazonia! - NSD1 Probe #219084_at; NSD2 Probe #209052_s_at; NSD3 Probe #218173_s_at - and five normal CD34+ samples included in the same databank). The following parameters were used to evaluate treatment outcome: complete hematological remission (CHR); 5-year Disease-Free Survival (DFS) and 5-year Overall Survival (OS) rates. Results: The median age of the learning set was 46y with 70 males (48%). NSD1, 2 and 3 expression levels are lower in de novo AML samples compared to CD34+ cells (P<.001). These results were not validated in an Amazonia! cohort, which patients with AML had a higher-than-normal expression of NSD1 and 3 (P=.01). Expression of all NSD was closely correlated in the learning set cohort (r>0.96) albeit only NSD2 and 3 had a correlation in the validation cohort (r=0.6). Baseline characteristics (sex, age, leukocyte count, cytogenetic risk, FLT3 and NPM1 mutations)were similar between the cohort of patients with NSDs low and high expression, except for higher blast count in BM, but not in peripheral blood, in high expression group for NSD1 and 2 (P=.021 and P=.033, respectively). Overall, 78/146 (57%) patients achieved CHR. Patients with a high NSD1 expression had a lower CHR rate (44%) compared with those a low expression (56%) (OR: 0.5; 95%CI: 0.2-0.9; P=.04). With a median follow up of 21 months (1-61 months) the estimated 5y DFS rate was 49% (95%CI: 30-65%). The group with high NSD1 expression presented a lower 5y DFS rate (13%; 95%CI: 1-42%) than those with low NSD1 expression (72%; 95%CI: 47-87%) (P=.003). This result was consistent with the multivariable proportional hazards analysis (HR: 7.3; 95%CI: 1.3-38; P=.02). The estimated 5y OS rate was 24% (95%CI: 14-35%) which patients with high NSD1 expression exhibiting lower 5y OS rate (13%; 95%CI: 4-29%). The median age of the external validation cohort was 58y with 107 males (62%). Only NSD2 levels had a association with higher diagnosis age (P=.008). With a median follow up of 40 months (1-118 months) the NSD expression may predict only a higher 5y OS rate in patients with high NSD2 expression (30%; 95%CI: 26-49%) retained the result in a multivariable model (HR: 0.53; 95%CI: 0.3-0.9; P=.02). Conclusions: In summary, NSD1 and 2 transcript levels were independent factors associated with treatment outcomes in two AML cohorts treated with standard protocols based on anthracyclines and cytarabine. Disclosures No relevant conflicts of interest to declare.
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